Glucocorticoids exert several effects in tissues which have receptors for them. They regulate the expression of several genes either positively or negatively and in a direct or indirect manner. They are also known to arrest the growth of certain lymphoid cells and in some cases cause cell death (1-5). Because of their ability to kill cells, glucocorticoids have been used for decades in the treatment of leukemias, lymphomas, breast cancer, solid tumors and other diseases involving irregular cell growth, e.g. psoriasis. The inclusion of glucocorticoids in chemotherapeutic regimens has contributed to a high rate of cure of certain leukemias and lymphomas which were formerly lethal (6). Although it is clear that glucocorticoids exert these effects after binding to their receptors, the mechanism of cell kill is not as yet understood although several hypotheses have been proposed. Among the more prominent hypotheses are: the deinduction of critical lymphokines, oncogenes and growth factors; the induction of supposed "lysis genes"; alterations in calcium ion influx; the induction of endonucleases and the induction of a cyclic AMP-dependent protein kinase (7-12).
The human glucocorticoid receptor is made up of 777 amino acids and is predominantly cytoplasmic in its unactivated, non-DNA binding form. When activated, it translocates to the nucleus. In order to understand the role played by the glucocorticoid receptor in the different cell processes, the receptor was mapped by transfecting receptor-negative and glucocorticoid-resistant cells with different steroid receptor constructs and reporter genes like CAT or luciferase which had been covalently linked to a glucocorticoid responsive element (GRE). From these studies, four major functional domains have become evident. From amino to carboxyl terminal end, these are: the tau 1, DNA binding, tau 2 and steroid binding domains in succession. The tau 1 domain spans amino acid positions 77-262 and regulates gene activation. The DNA binding domain is from amino acid positions 421-486 and has nine cysteine residues, eight of which are organized in the form of two zinc fingers analogous to Xenopus transcription factor IIIA. The DNA binding domain binds to the regulatory sequences of genes that are induced or deinduced by glucocorticoids. From amino acids 532-555 is the tau 2 domain which is the second domain important for transcriptional activation. Towards the carboxyl terminal end, from amino acids 555 to 777, is the steroid binding domain. This domain binds glucocorticoid to activate the receptor. This region of the receptor also has the nuclear localization signal. Deletion of this carboxyl terminal end results in a receptor that is constitutively active for gene induction (up to 30% of wild type activity) and even more active for cell kill (up to 150% of wild type activity) (13-26).
From previous experiments, the present inventors have shown that an intact GRE-specific DNA binding domain of the glucocorticoid receptor and its flanking sequences comprising in total, amino acids 1-8 connected to 386 through 532 is sufficient for the cell kill function of the receptor (27, 28). It was also shown that amino acids 1-465 were sufficient for the lethal function. Hollenberg et al. (14) describe constructs with NH.sub.2 terminal deletions that lose activity for gene induction; and Dieken and Miesfeld (42) found that the amino terminal domain was required for cell kill. Thus the existing data indicated that at best some part of the amino terminal domain as well as the DNA binding domain were essential. A large additional body of published data has mapped many important functions of the receptor (nuclear translocation, heterologous protein-binding, ligand-binding, trans-activation) and thus its critical function required these domains (2-4, 18).
The present invention identifies a short sequence less than 100 amino acids in length, and not predicted from prior work, which is lethal in a cell line of T lineage. This sequence codes for only 23 amino acids preceding the DNA binding domain, and stops the domain short due to a missense mutation in the second zinc finger. The resulting predicted peptide replaces the normal carboxy-terminal end of the DNA binding domain with a missense sequence of 21 amino acids. Such a peptide could not be predicted, on the basis of published knowledge, to have any biological function and potency equal to that of holoreceptor plus steroid. Use of this construct in chemotherapeutic regimens could circumvent the need for steroid treatment in glucocorticoid-resistant malignant cells. When properly targeted towards a proliferative cell it may be used to cure several forms of proliferative disease.